1.1 INTRODUCTION

Animal husbandry plays an important role in Indian agriculture. Indians by

large are vegetarian and as such the only source of animal protein is milk and milk

products. With the increasing population not only food security but also nutritional

security is required. In this context milk is valuable part of our nutrition. The total

population of cows in India was approx. 199.1 million and the total buffalo

population was 105.3 million (18th

Livestock Census, Department of Animal

Husbandry, Dairying and Fisheries, Ministry of Agriculture, 2007). But as per 19th

livestock census (19th

Livestock Census, Department of Animal Husbandry,

Dairying and Fisheries, Ministry of Agriculture, 2012) the total cow population

reduced to 190.9 million. This shows an overall decline of 3.33% in the total

livestock population as compared to 18th

livestock census. Cattle reared in India are

low producers. On an average the native cow produces 2-3 litres of milk and buffalo

milk is hardly on an average 7 or 8 litre per day, whereas cross bred cows produce

15- 30 litres of milk per day. During the last decade the dairy industry has evolved

towards intensive milk production system to fulfil the demand of the increasing

human population. Hence, large numbers of high yielding exotic cattle (Holstein

Friesian) are being imported under various integrated development projects

throughout the country. Since 70’s the massive cross breeding programme with the

semen of high yielding cows of American and European origin has been taken up.

The 20% of cattle population, approximately 39.82 million are cross bred cows.

There are many cross bred cows which are producing over 5000 litre of milk per

lactation. Thus the milk production has initiated jobs, income and foreign exchange

generation. This is likely to pick up. However, cross bred cows are very susceptible

to heat stress and blood protozoans whereas, the native cows are somewhat resistant.

Cross bred cows are sensitive to hot and humid climate. The impact of heat stress on

dairy cows is measured by calculating the Thermal humidity Index (THI). It is used

to evaluate the climatic conditions that contribute to heat stress [1, 2]. THI is used

for estimation of the magnitude of the heat stress by using ambient temperature and

relative humidity [3]. Theileriosis and babesiosis are important blood protozoan

diseases in cattle that affect the economy of country. Both diseases are transmitted

by ticks [4]. Theileria and Babesia both have same symptoms like high fever and

both are fatal diseases. In babesiosis blood comes out with the urine and hence it is

also known as Red water disease. Theileria vaccines are not easily available as they

are very costly and these vaccines also require liquid nitrogen for its preservation.

Theileriosis is a vector borne disease and transmitted by Hyalomma ticks.

Different Theileria species are found all over the world in cattle. Arnald Theiler first

reported the Theileria parasite [5]. Theileria can exist in erythrocytes and

lymphocytes of their host. The taxonomy of Theileria species are based upon

morphology and geographic area [6]. The two known pathogenic species of

Theileria in cattle are T. parva and T. annulata [7], which cause East Cost Fever and

Tropical Theileriosis respectively in tropical and subtropical countries [8]. The

mortality rate due to T. annulata is 70-80% while due to T. parva, 100% mortality

rate has been recorded in exotic cattle [9]. In calves high mortality has been

observed as compare to adult cattle, however if adult cattle survives after infection it

becomes immune [10]. In introduced exotic cattle 30 to 90% mortality rate has been

observed due to Theileria but in indigenous breed mortality rate is either 5% or less

[11]. The Sahiwal breeds of cattle are highly resistant to ticks, which are responsible

for transmission of Theileria [12]. However the cross bred cows suffer from blood

protozoans which are also known as tick borne infections. Exotic livestock kept

even at low tick challenge are highly susceptible [13, 14]. If introduced exotic cattle

are kept in the same herd with indigenous breeds, the chances of parasitic infection

increases [9]. Babesia is limited to adult animals whereas Theileria affects all ages

of cow and it can be transmitted to new borne calf through mother. The total

number of death by theileriosis may be 80-90%, if not treated.

Theileria annulata (T. annulata) causes tropical theileriosis (also

known as tropical piroplasmosis, Egyptian fever or Mediterranean Coast fever) in

cattle with a mortality rate from 10-90% [6]. T. annulata is vectored by Hyalomma

ticks, which become infected as a larva or nymph and infection is then passed on by

the nymph or adult, respectively [6]. In the first step of life cycle of Theileria,

sporozoites present in the salivary gland of tick are transmitted into the host with

tick saliva while feeding and these sporozoites infect the lymphocytes. Infected

lymphocytes start dividing and schizonts (also known as Koch’s Blue Bodies) are

formed inside the infected lymphocytes converting into the merozoites. These

merozoites come out from lymphocytes and enter into the erythrocytes where they

are known as piroplasms [15]. Two forms of piroplasms have been observed in

erythrocytes; either they are slender comma shaped or spherical ovoid form [15].

The infective stage is piroplasm stage which is taken by ticks while taking a blood

meal from an infected host. After attachment of infected ticks, the first clinical sign

of theileriosis appears within 7 to 15 days in cattle. The body temperature increases

to 42° C [16, 17]. Anaemia develops in the infected animal followed by loss in

health condition. Other clinical signs observed in animals due to theileriosis are

lacrimation, corneal opacity, nasal discharge, terminal dyspnea, diarrhoea and

sometimes haemoglobinuria is also seen.

Cattle infected with T. annulata are generally diagnosed by examining

Giemsa’s-stained lymph node biopsy smears and blood smears for the detection of

macroschizonts and piroplasms respectively [18]. The morphology of piroplasms of

Theileria species is more or less similar so it becomes difficult to differentiate them

on blood slides by microscopic examination [19]. Serological tests like indirect

immunofluorescent antibody test (IFA) are used for detecting antibodies present in

the blood of the infected animal [19]. However, IFA test is not successful to detect

all infected cattle especially carrier cattle. It also has a drawback i.e. cross-reactivity

within closely related Theileria species, which limits the specificity of serological

tests [20]. Furthermore in carrier animals the antibodies disappear after a period but

Theileria piroplasms remain present in low numbers. Therefore, carrier animals are

not diagnosed by this serological test and they are still capable of infecting ticks

[21]. The 18S small subunit ribosomal RNA gene is a highly conserved region in

the nuclear genome. The small subunit ribosomal RNA gene (SSUrRNA) has high

levels of conservation so it is useful for phylogenetic analysis [22]. The small

subunit ribosomal RNA gene of T. annulata was compared to SSUrRNA gene

sequence of Apicomplexans, Dinoflagellates and Ciliates and a close phylogenetic

relationship was observed between Apicomplexa and Dinoflagellata [23]. To

differentiate six Theileria species, primers have been designed for small subunit

ribosomal RNA gene and it is useful for identification of Theileria infected animals

as well as carrier animals [24]. Through microscope only 50% animals can be

diagnosed [25]. However PCR based diagnostic test can detect the positive clinical

and sub clinical cases. If diagnosed positive, the drug Buparvaquone if given in time

can save the animal. But Buparvaquone doesn’t give 100% effective results as it is

effective only if animal is treated in early stage of infection. In the severe stage of

disease it failed to cure the animal [26]. The parasite has the detrimental effect on

the cows, it causes high mortality and the animals that survive have irreversible loss

of milk production and reproduction.

In carrier animals piroplasms persist in their blood at very low number

but they are sufficient to infect the ticks and are not diagnosed easily by the routinely

used diagnostic techniques [25]. In the sub clinical cases, the parasite is in the lymph

gland and it reduces the production and upset the reproduction. The PCR test once

settled and equipment is available, costly animals can be saved by using this

technique and increase production and reproduction. Nonimmune cattle that are

imported from the places where Theileria is prevalent are susceptible to disease.

Theileria infection is a major issue for the livestock development programmes in

many places. Cattle of European origin like Holstein Friesian (Bos taurus) are very

much susceptible to Theileria, thus it is a big issue for cattle farming [27]. Seasonal

activity of Rhiphicephalus appendiculatus was studied in Zimbabwe and Rhodesia

and it was found that the adults are highly active in rainy season and larval and

nymphal are highly active in dry season [28]. They also found that activity was

regulated by influence of temperature, humidity and dry length.

Theileria is usually thought as disease of warmer climate and has not

been reported in Uttarakhand to the best of our knowledge. Earlier studies suggested

that this disease did not occur in the Himalayan regions [29]. It was thought that in

Uttarakhand haemoprotozoans were not prevalent. As no sensitive diagnostic

method for detection of clinical cases or carrier animals was in use and also no cost

effective treatment was available for theileriosis, many of diseased animals were left

untreated.

Dehradun district of Uttarakhand is located at the foothills of Himalayas.

Dehradun is situated between latitudes 29 °58' N and 31°2'N and longitudes 77° 34'

E and 78° 18'E. The district is bordered by hills, the Himalayas in the north and the

Shivalik Hills to the south. The district is also bordered by the rivers, Ganges to the

east and Yamuna to the west. The physical geography of Dehradun district varies

from Himalayan Mountains to Plains. The spring here spans from March to mid

April. Summer follows the spring season from mid April to June end. Monsoon

starts after June and ends in October. By November the winter starts. Transportation

of animals occurs here mainly from Haryana, Punjab. In Dehradun district

particularly in Dehradun city the cost of milk is more than the cost of diesel, whereas

the fodder production is scanty, not much green are available, and cattle are kept on

bhusa. There is a great paucity of land. So there is no space for grazing calves. As

such the high producing animals producing over 30 litre of milk per day are kept for

making good profit. There is no room for low producers. These high producing

animals are mainly crosses of Holstein, which are prone to heat stress and blood

protozoans like Theileria.

Figure 1.1: Map of Dehradun showing Border States and black arrows show

locations of sampling (Source: www.slbcuttarakhand.org.in/images/dehradun.jpg)

Uttarakhand is a relatively new state and many cross bred cattle were

introduced to boost the milk yield of the state. As Cross-bred cattle of Holsteins are

high milk producers so they are introduced in Uttarakhand, especially in Dehradun

region from the neighbour states like Haryana, Punjab etc. where these diseases are

prominent and many of these animals may be carriers. These carrier animals are the

source of infection. Infection is usually transmitted by ticks that migrate from

carrier animals to non-infected animals.

Despite all efforts, the milk yield of dairy in Uttarakhand is comparatively

low. The problem of heat stress in dairy cattle has recently increased due to

anticipated increase in environmental temperature by global warming. Heat stress is

very important stress factor and it cannot be indicated only in terms of temperature.

When high humidity level coincides with high temperature, it results in oppressive

and stressful conditions. The THI is measured by combining temperature and

humidity both and has been used to quantify heat stress in dairy animals.

Temperature humidity index (THI) was originally developed for humans

and extended to cattle. High yielding cross bred cows are more sensitive to hot and

humid environment than low yielding cows while the native cattle are well adapted

to these environmental conditions. “The higher susceptibility of high yielding dairy

cattle to heat stress could be explained by the substantial increase in metabolic heat

output these cows are experiencing due to the high milk production, which may

exacerbate the problem of heat stress in high yielding dairy cows” [30]. The exotic

Holstein crosses are uncomfortable above the temperature of 25°C. When Heat

Stress Index for Cattle is 71 or below, cattle are in the zone of comfort i.e. 72 is

optimum, below this animals are in zone of comfort. Values ranging from 72-80

indicate “Mild Stress” and from 81- 90 indicate “Medium Stress”. When Heat Stress

Index values goes above 90 it indicates “Severe Stress”.

Heat stress makes many physiological as well as behavioural changes in

animal’s body like less feed intake, decline in milk production, reduce growth,

reduction in reproduction activity, increase in respiratory rate and rise in body

temperature. Hormones in plasma are potential indicator of the physiological status

of a cow. Environmental temperature, especially high temperature, depresses the

thyroid function. In heat stressed animals, 25 % lower concentrations of T3 and T4

in blood plasma were observed. It takes several days for T3 and T4 level to return at

normal range. The readjustment in thyroid response is chronic and as a result,

summer thyroid activity remains low with decreased metabolic rate, reduced feed

intake and growth and low milk production under heat stress [31].

Figure 1.2: Thermal Humidity Index chart showing different levels of heat stress

at different temperature and relative humidity (Source: www.extension.umn.edu)

This study was aimed to assess the occurrence of Theileria in district

Dehradun of Uttarakhand using sensitive molecular diagnostic technique polymerase

chain reaction (PCR) based diagnostic tool and optimization of thermal humidity

index (THI). This DNA technology monitors the presence of parasites, which

otherwise are not visible by microscopic examination and in preclinical cases.

1.2 STATEMENT AND GENESIS OF THE PROBLEM: -

The Graphic Era University in Dehradun district maintains a herd of 70

cattle and supplies milk. It is also a source of vermicompost. In 2010 some cows fell

sick, few cows were treated with antibiotic and three cows died. The blood

examination at IVRI and at this lab revealed that these animals were suffering from

theileriosis which mainly attacks high yielding cross bred cows but not the native

cows. Six cows added from Rajasthan were diagnosed carriers hence affecting the

cow population. Microscopic examination of blood smear did not reveal all positive

cases as were as the carrier cases. So the molecular diagnostic tool, polymerase

chain reaction test was applied to diagnose the true status of the disease. Further the

same was applied to different herds in the Dehradun district.

To increase the milk production in Uttarakhand many dairy farms have

imported high yielding exotic cattle. However milk production usually declined in

most of the dairies. Some cows fell sick and some even died. Hence this study was

planned to investigate the possible reason for decrease in milk production focussing

on blood protozoans and heat stress.

1.3 OBJECTIVES: -

The aim of the study was early diagnosis of theileria by polymerase

chain reaction in crossbred cows in Dehradun, Uttarakhand. The study has been

focused on Uttarakhand with the following objectives-

1. To check/reduce the mortality in cows by diagnosis of blood protozoans.

2. To check the carrier animals (preclinical cases of Theileria).

3. To know the species of Theileria.

4. To find out correlation between Theileria, Thermal Humidity Index (THI) and

milk production.

5. To study hormone profile to monitor the degree of stress.

1.4 ORGANIZATION OF THESIS

The thesis has been presented in 7 chapters:

Chapter 1 deals with the introduction and motivation of the research problem.

Livestock production is very important for the improvement in agriculture of India.

Indians by large are vegetarian and as such the only source of animal protein is

milk and milk products. Cattle reared in India are low milk producers. Hence large

numbers of high yielding exotic cattle are being imported throughout the country.

There are many cross bred cows which are producing over 5000 litres of milk in

365 days per lactation. But cross bred cows are very susceptible to heat stress and

blood protozoans, which may cause 90 % mortality and also lowered milk

production. The native cows usually do not suffer from Theileria infection.

Haemoprotozoans pose a serious challenge to the exotic cross bred cattle

population. Tropical theileriosis is a disease found in cattle, caused by T. annulata

and is transmitted through Ixodid ticks of genus Hyalomma. This disease has not

been reported from the Himalayan regions as the temperature is not favourable for

the survival of ticks. Earlier it was thought that Uttarakhand was free from blood-

borne parasites and their vectors. As no sensitive diagnostic method is available

and in use for detection of clinical cases and carrier animals of Theileria and also

no effective treatment within reach of small dairy holders is available for

theileriosis, thus resulting in maximum cases left untreated. Carriers are the source

of infection and aid in spread of the disease. Hence this study was aimed at

assessment of extent of spread of this disease in district Dehradun of Uttarakhand.

The study also aimed at monitoring the heat stress through Thermal Humidity

Index (THI) and monitoring the blood protozoan by microscopic examination and

by Polymerase Chain Reaction (PCR) which is a recent DNA Technology. This

DNA technology monitors the presence of parasites which is not visible by routine

microscopic examination and in preclinical cases.

This critical review on Theileria parasite is presented in chapter 2. Theileria is

intracellular protozoan parasite. The Theileria parasite was first reported by

Arnald Theiler. “Theileria are small round, ovoid, irregular or bacilliform shaped

parasites”. T. annulata was first described in 1904 in Transcaucasian cattle [32]. It

usually infects exotic and cross bred cattle. This chapter reviews history,

epidemiology, etiology, symptoms, diagnosis and control of Theileria. It also

includes reports of theileriosis from different regions of India. Ticks act as

biological vectors for the transmission of Theileria species. Rhipicephalus

appendiculatus and Hyalomma ticks are the most important vectors for the

transmission of T. parva and T. annulata respectively. Female ticks are more

responsible for the transmission of Theileria parasite than male ticks. Many cows

are apparently healthy but they are carrier of Theileria. Carrier means they harbour

parasite and can transfer disease in cows which are not carriers. The carrier

animals have developed antibodies and these animals do not suffer from Theileria

again but piroplasms remain in their blood in very low number so such carrier

animals can’t be diagnosed by microscopic examination. However the production

of milk and reproduction of such animal is affected. Carrier animals are having

low numbers of infected erythrocytes infect ticks and play major role in

transmission of the disease through ticks. Therefore, by identifying the carrier

animals the spread of infection can be controlled to some extent. However,

detection of carrier animals is not easy by microscopy as they have very low

number of infected erythrocytes, and also to differentiate pathogenic species from

the non-pathogenic species of Theileria is very difficult by microscopic

examination. Although serological tests can also be used as effective diagnostic

tools by detecting antibodies but these tests are also not 100 % accurate due to

cross-reactivity of antibodies of other species [33]. In carrier animals antibodies

disappear with time but piroplasms may reside in very low number, therefore these

animals show negative serological tests and can still be able to transmit the disease

by ticks. Several molecular diagnostic procedures like PCR were developed for the

identification of these parasites [34]. If animal suffering from tropical theileriosis

are treated with antiparasitic drugs, Theileria annulata does get removed from

lymph nodes but remains in blood in very low number [26]. As DNA sequence of

the parasite is known and PCR is available, parasites can be detected within blood

samples of the infected animal. PCR has become the sensitive and accurate

diagnostic technique for the detection of T. annulata [35]. PCR enables us to

detect parasitic infection with clinical or without clinical signs [21]. Exotic cattle

are also susceptible to heat stress which is measured by thermal humidity index.

This chapter discusses the heat stress, factors responsible for heat stress, effect of

heat stress on cattle milk yield and thyroid hormone.

In chapter 3 Theileria has been monitored by using microscopic examination

technique. This chapter documents the detection of Theileria in cross bred cattle

by microscopic examination. On the basis of information collected from different

areas of Dehradun district that cows are dying and not responding to antibiotic and

usual treatment, the animals were screened for blood protozoan i.e. Theileria which

was not prevalent in Garhwal region of Uttarakhand. Blood sampling was done

from different locations of Dehradun district. Blood sampling procedure was

approved by Institutional Animal Ethical Committee (IAEC). Screening of the

animals was done by blood smears examination. The blood smears were examined

microscopically using Giemsa stain to detect the occurrence of Theileria. For

knowing probable months of the occurrence of Theileria the study was based on

season-wise screening of the animals. As per the preliminary survey, in different

places of Dehradun like Raipur, Subhashnagar etc. some cases of theileriosis were

diagnosed and some carrier animals were established. In this survey 20 positive

cases for Theileria out of 58 animals were observed. In the experimental study,

301 cattle were screened for the prevalence of Theileria and 82 (27.2%) were

found positive for Theileria by microscopic examination. Highest prevalence was

found in the rainy season with prevalence rate of 45.4% followed by summer with

27.4%. In spring season and winter season the prevalence rate was around 9% and

8.8% respectively.

Chapter 4 describes the monitoring of theileriosis and carrier cases by using the

polymerase chain reaction (PCR) test. The clinical and subclinical cases or carrier

cases were subjected to molecular diagnostic tool i.e. polymerase chain reaction.

All the samples were examined by PCR test using Theileria specific primer set

989/990 [989(F) AGTTTCTGACCTATCAG and 990(R)

TTGCCTTAAACTTCCTTG] for the SSU rRNA gene. Identification of Theileria

species was made on the basis of polymerase chain reaction based test. PCR

positive samples were further diagnosed by using T. annulata specific primer set

N516/N517 [N516 (F)-GTAACCTTTAAAAACGT3’ and N517 (R) –

GTTACGAACATGGGTTT]. This is a step forward in saving exotic cattle and

making it possible for maintaining exotic cattle for increasing milk production.

Thereby it is a thrust area for generation of employment and economy for masses.

By using PCR besides 82 microscopically positive samples, 16 other samples were

also detected as positive for theileriosis. These cattle were categorized as “carrier

cattle”. So Theileria genus specific PCR could detect 98 samples (32.5%) as

positive. Hence PCR technique is more sensitive and accurate.

Chapter 5 discusses the effect of heat stress in cross bred dairy cows and its

correlation with milk production. To measure the heat stress, the daily temperature

and humidity variation for three years were continuously monitored. With the help

of these two parameters we calculated the thermal humidity index (THI). To

assess the effect of heat stress on milk production 40 cattle were observed. These

cattle were divided into two groups i.e. high yielding cattle which were imported

from other states and low yielding cattle which were maintained here for the last

two years. The daily milk production of all the cattle was measured to verify the

effect of heat stress on milk yield. As hormones are the good indicator of stress,

we monitored thyroid hormones (T3 and T4) to assess the effect of heat stress on

hormone concentration in serum of high yielding cattle by using the enzyme linked

immuno-sorbent assay (ELISA) test kits and then correlated it with the thermal

humidity index by plotting a graph. The THI rose above 72 in the month of July,

till October and in November it decreased. In high yielding cows, once the milk

production declined it did not rise. Even after the THI declined there was loss of 8-

9 litres of milk per cow everyday and this loss was irreversible. The low yielding

cows, producing less than 8-10 litre of milk, were not much affected by increase in

thermal humidity index or stress level. As THI increases, milk yield decreases.

This decrease in milk production can range from 40- 44%. The thyroid hormones

level was also observed to be decreased in the summer season when the thermal

humidity index was in stress zone i.e. above 80. When THI value was 72 or below

72, the thyroid hormone levels either increased or remained in normal range.

Chapter 6 deals with the general discussion part of the entire study. Theileriosis is

a fatal parasitic disease which is seasonal usually starts from the month of May and

its occurrence increases gradually till rainy season. The highest prevalence of

Theileria was found in the months from August to October as the highest

abundance of the ticks was reported in the months of June-July. Carrier cattle

(infected but not showing symptoms) are usually the chronic carriers of

piroplasms. They become the source of infection when carrier cattle are reared

together with healthy cattle. Ticks from carrier cattle act as vectors and transmit

the disease to healthy cows (cross bred cows, like Holstein Friesian, are more

susceptible to infection). Thermal humidity index was also found high in the

months from August to October i.e. above 80. This also causes distress in the

animals and lowered the milk production especially in the high yielding cows.

However the low milk producers were not much affected by increase in thermal

humidity index or stress level. In this connection the thyroid hormone levels were

monitored which were found very high during winter months and low in summer

months. Low thyroid hormone level in the summer months also shows the degree

of heat stress in animals.

Chapter 7 concludes the study and recommends the future prospects of the study.

This is one of the first reports documenting spread of Theileria in Himalayan

region of Garhwal, Uttarakhand. It was concluded that there is outbreak of

theileriosis in Dehradun district. Diagnosis of Theileria is done by polymerase

chain reaction test. This technique is very sensitive and detects carrier animals.

Highest prevalence of Theileria was observed in rainy season when the thermal

humidity index was above 80. As the disease is transmitted from the carrier

animals introduced from Theileria endemic areas of the country, before their entry

they must be quarantined. The high milk producing animals were highly affected

by heat stress. Decrease in thyroid hormone level also showed that the animals are

affected by heat stress. This chapter also discusses the future prospects of the

study. Theileriosis in Dehradun is spreading at a faster rate which is to be tackled

immediately.